Biology subjects

Lindau, M.

Publications and source records attributed to Lindau, M..

3 recordsLinked to original sources

bioRxiv2026

Electrostatics and Local Aromatic Residues Govern Lipid Binding and Membrane Penetration of Synaptotagmin C2 Domains

Synaptotagmins (Syts) are Ca{superscript 2}-sensing exocytosis regulators whose tandem C2 domains interact with phosphoinositides and membranes to trigger neurotransmitter and hormone release. Although Ca{superscript 2} binding is known to enhance C2 domain-membrane interactions, the sequence determinants governing lipid binding and membrane penetration across Syt isoforms remain incompletely understood. Here, we performed MARTINI coarse-grained molecular dynamics simulations of isolated C2A and C2B domains from eight Ca{superscript 2}-sensing Syt isoforms (Syt1, Syt2, Syt3, Syt5, Syt6, Syt7, Syt9, and Syt10) interacting with phosphatidylinositol 4,5-bisphosphate (PIP2)-containing plasma membranes. To systematically modulate electrostatic properties, we introduced partial and full charge-flip mutations at conserved acidic residues within the calcium-binding loops (CBLs). By integrating simulations across multiple isoforms and charge states, we sought to identify the dominant sequence determinants governing membrane interactions. We found that PIP2 binding to both, CBLs and polybasic patches (PBs), is associated with loop net charge, yielding correlations > 0.95 across all isoforms. However, membrane penetration is not sufficiently explained by loop net charge alone. The local phenylalanines additionally increase membrane penetration independent of loop net charge. Together, these findings establish a comprehensive electrostatic-aromatic framework where loop net charge governs PIP2 binding, whereas loop net charge and local phenylalanine enrichment jointly govern membrane penetration across Syt C2 domains.

biophysics↗

bioRxiv2025

Syntaxin 1A Transmembrane Domain Palmitoylation 1 Induces a Fusogenic Conformation

Neurotransmitter release is triggered by the fusion of synaptic vesicles with the plasma membrane, orchestrated by SNARE proteins Synaptobrevin 2 (Syb2), Syntaxin 1A (Stx1A), and SNAP25. Recent experimental studies showed that Stx1A palmitoylation of C271/C272 promotes spontaneous neurotransmitter release. However, the mechanistic role of SNARE transmembrane domain (TMD) palmitoylation in membrane fusion remains unclear. To investigate the structural and functional implications of TMD palmitoylation, we employed coarse-grained molecular dynamics simulations with the MARTINI force field. In simulations of individual SNAREs and of SNAP-25/Stx1A (t-SNARE) complexes in a membrane the palmitoyl chains of Syb2 and Stx1A localize to the membrane midplane, with Stx1A palmitoyl chains bending toward the extracellular leaflet. Non-palmitoylated Stx1A assumed a conformation where the SNARE domain was lying flat, adhering to the intracellular surface of the membrane. Stx1A dual palmitoylation induced dramatic changes, reducing the tilt of its TMD and stabilizing a more upright conformation of its SND. This conformation resembles the Stx1A conformation in a s Stx1A-SNAP25 t-SNARE complex, providing a potential mechanistic explanation of how Stx1A TMD palmitoylation facilitates early steps in SNARE complex formation and thus promotes spontaneous release. In simulations of the late steps of layers 5 to 8 SNARE complex zippering in a system of 4 SNARE complexes bridging a 10-nm nanodisc and a planar membrane, FPs spontaneously opened after a few hundred nanoseconds, preceded by distal leaflet lipid transfer and followed by FP flickering conductance before FP closure. At this stage, Stx1A TMD palmitoylation delayed lipid transfer and FP formation and decreased FP flicker open times, whereas the palmitoylation of Syb2 did not affect fusion pore dynamics. These findings suggest that after facilitation of priming before FP opening, Stx1A TMD palmitoylation, directly affects FP dynamics. These results highlight the essential role of SNARE TMD palmitoylation at multiple stages of neurotransmitter release. Statement of SignificanceSynaptic vesicle fusion is critical for neurotransmitter release, enabling neuron-to-neuron communication at synapses. Post-translational modifications, such as palmitoylation, are known to influence this process. Using MARTINI coarse-grained molecular dynamics simulations, we examined the impact of SNARE transmembrane domain (TMD) palmitoylation on SNARE protein conformation and fusion dynamics. Stx1A palmitoylation reduces its TMD tilting and changes its SNARE domain conformation, facilitating SNARE complex formation. In fusion pore (FP) simulations, Stx1A palmitoylation delayed FP opening, decreased FP flicker open times, and shortened FP conductance flicker durations by direct interactions with the FP. Interestingly, dual palmitoylation of Stx1A and Syb2 restored flickering duration but decreased FP opening probability within 4 s, suggesting a nuanced role of TMD palmitoylation in modulating neurotransmitter release.

biophysics↗

bioRxiv2025

SNARE complex assembly and disassembly dynamics in response to Ca2+ current activation in live cells

A SNAP25 based FRET construct named SCORE (SNARE COmplex REporter) has revealed a transient FRET increase that specifically occurred at fusion sites preceding fusion events by tens of milliseconds and presumably reflects vesicle priming. The FRET increase lasts for a few seconds until it is reversed. In those experiments, the FRET increase was found to be localized to areas <0.5 {micro}m2 at sites of transmitter release as detected amperometrically using electrochemical detector arrays. Due to the localization to such small areas, it was unknown if the reversal of the FRET increase is due to local dispersion of high-FRET SCORE copies leaving the site after fusion and exchange with surrounding low-FRET copies, or if it reflects disassembly of the high-FRET complexes. To resolve this question, we performed whole-cell patch clamp pulse stimulation experiments, imaging the entire footprint of the cells in Total Internal Reflection Fluorescence (TIRF) excitation mode such that diffusional exchange between high-FRET and low-FRET copies does not produce a net FRET change. We show here that pulse stimulation of calcium currents results in FRET ratio transients with a time course very similar that related to fusion events. By comparing the kinetics of the FRET ratio decay with analytical and numerical diffusion simulation results, we show that the experimentally observed kinetics cannot be explained by diffusional exchange and conclude that the SCORE FRET ratio transients reflect incorporation of SCORE in SNARE complexes followed by SNARE complex disassembly. Experiments using Synaptobrevin 2/Cellubrevin double knock-out mouse embryonal chromaffin cells showed no pulse induced FRET change, indicating that the vSNARE is required for the incorporation of SCORE (or SNAP25 in wild type cells) in the SNARE complex during priming. Statement of SignificanceIn chromaffin cells, SNAP25-based FRET constructs (SCORE) revealed transient FRET increases within <0.5 {micro}m2 areas, preceding individual fusion events that reversed within seconds. It remained unknown whether this reversal stems from high-FRET complex disassembly or diffusion-mediated exchange with low-FRET complexes. Here, we performed whole-cell patch-clamp pulse stimulation with TIRF microscopy, imaging large [~]30 {micro}m2 areas of the cell footprint. Calcium currents induced FRET transients with the same decay time constant of [~]1.5 s, significantly shorter than the time scale of diffusion. The SCORE FRET ratio thus reports in real time the dynamics of SNRE complex assembly and disassembly in live cells. Using Synaptobrevin 2/Cellubrevin double knock-out mouse chromaffin cells we show that vSNAREs are required for SNAP25 incorporation into SNARE complexes during priming.

biophysics↗